Journal of clinical virology plus最新文献

Background

Very limited information is available on SARS-CoV-2 seroprevalence in infants in sub-Saharan countries.

Objective

In this study, we aimed to determine the rate and the temporal evolution of SARS CoV-2 seropositivity in breastfed Malawian infants.

Study design

Blood samples (n = 250) from 158 infants, born to HIV-negative women and women living with HIV, collected from February 2020 to May 2021, were first tested using an Anti-IgG/A/M SARS CoV 2 ELISA assay against trimeric spike protein, and then, if positive, confirmed using a second ELISA assay detecting IgG against Receptor Binding Domain.

Results

The confirmed prevalence of anti-SARS CoV-2 antibodies was 31.0% (95% CI: 23.7%-38.3%) with no significant difference between HIV-exposed and HIV-unexposed infants (29.3% and 37.1% respectively, P = 0.410). The presence of anti-SARS-CoV-2 IgG was not associated with maternal socioeconomic or demographic indices.

Conclusions

Our data underline the wide spread of the SARS-CoV-2 infection in the pediatric population in sub-Saharan Africa. Design of more specific serological tests for African samples and improvements in serosurveillance programs are needed for more rigorous monitoring of the dynamics of SARS-CoV-2 infection in Africa.

Background

Improving the early identification of HIV-1-infected newborns with birth testing is critical to comprehensive early infant diagnosis and care for newborns living with HIV-1. Automated RNA quantification systems are valuable diagnostic tools, but the volume of plasma that viral load platforms require makes their widespread use for young children difficult.

Method

Seventy-nine plasma samples with different viral load ranges were evaluated in parallel with the use of 1x PBS, pH 7.4, to supplement the required volume at dilutions factors from 1:2 to 1:50. Viral load quantification assays were evaluated using ABBOTT Molecular platforms, USA.

Results

Using 1x PBS, at 1:10 dilution (70 µL plasma in 630  µL 1x PBS), a sensitivity of 100% and 100% specificity were obtained for detecting a viremia above 400 copies/mL (Kappa of 0.96, p < 0, 0001) for 1:50 dilution the sensitivity was 96% and the specificity 100% (kappa 0.90, p < 0.0001).

Conclusions

Although with reduced sensitivity, proportional to the dilution factor, the use of plasma does not influence the specificity of the test and allows the diagnosis of HIV-1 infections. Cases with very low viremia, a situation that may occur due to the treatment or prophylaxis of the mother and/or child, may go unnoticed with this procedure, and undiluted testing may be necessary.

Diagnosis of respiratory viruses traditionally relies on deep oropharynx or nasopharynx swabs collected by healthcare workers (HCW). However, outpatients must make an appointment, and the procedure can cause discomfort in patients. Self-collecting has the potential as a strategy to improve participants’ willingness to participate in diagnostics, surveillance, or studies.

We compared self-collected gargle fluids and nasopharyngeal swabs as a strategy for molecular diagnostics of respiratory viruses and compared the average cycle threshold (Ct)-values with those of samples collected by HCW. The study was conducted among technicians of the Laboratory of Clinical Microbiology and Infectious Diseases, Zwolle, the Netherlands, and their family members, between April 2019 and March 2020. It included a questionnaire regarding the severity and date of first symptoms and an assessment of the sampling experience. The primary outcome was the mean Ct of positive PCRs. Similar mean Ct values were obtained using self- or HCW-collected swabs. In addition, gargle fluids and self-swabbed specimens had comparable detection rates of respiratory viruses. Notably, most participants preferred gargling over self-swabbing. Interestingly, but not surprisingly, the time between the onset of symptoms and sampling was shorter in PCR-positive compared to PCR-negative participants.

Though this study was abrogated by the SARS-CoV-2 pandemic, the results indicate that both self-swabs and gargle fluids are acceptable for diagnosing common respiratory viruses in the outpatient population, including influenza virus, rhinovirus, adenovirus, SARS-CoV-2 and endemic human coronaviruses. Gargling could be considered an alternative sampling strategy and may enhance willingness to participate in screenings or diagnostics for respiratory viruses.

Background

Herpes simplex virus (HSV) seroprevalence in Bulgaria is higher than that in other countries but there is no information concerning involvement of these viruses in neuropathology. The aim of the present study was to determine the frequency of HSV DNA detection in cerebrospinal fluid (CSF) of patients with neurological diseases.

Methods

This study is a retrospective survey of test results obtained from January 2015 to October 2019. During this period 617 CSF specimens were tested for the presence of HSV-1 and/or HSV-2 DNA by PCR.

Results

Of all 612 CSF samples tested for HSV-1, 16.5% were positive. Of 547 CSF samples tested for HSV-2, 6.2% were positive. Co-infection with HSV-1 and HSV-2 was detected in 1.3% of tested 543 CSF samples. The difference of HSV-1 and/or HSV-2 prevalence in CSF of patients according to the gender and age was not statistically significant. The highest HSV-1 prevalence (25%) was detected in CSF from patients with multiple sclerosis (MS), followed by patients with encephalitis (20.6%). The highest HSV-2 prevalence (22.2%) was detected in CSF from patients with myelitis, followed by patients with encephalopathies (7.5%).

Conclusion

Our results show a considerable prevalence of HSV-1 and HSV-2 in CSF of patients with neurological diseases indicating the important role of these viruses in neuropathology in Bulgaria. HSV-1 is with predominant role in development of both encephalitis and meningitis in different age groups. Testing of CSF for HSV should be indispensable part of diagnostic algoritm of these diseases.

Respiratory Syncytial Virus (RSV) is one of the most common respiratory viruses causing acute respiratory tract infections (ARTI) in children. Detailed data on RSV infections including the RSV types circulating in Sri Lanka are not available. This study aimed to determine the prevalence, patterns and characterization of RSV associated ARTI in hospitalized children less than 5 years in a general hospital in Sri Lanka. We tested 500 nasopharyngeal aspirate (NPA) samples collected from children with suspected viral ARTI from May 2016 to July 2018 from Kegalle General Hospital, Sri Lanka for RSV using antigen detection by an immunofluorescence assay (IFA). RSV positive samples were further characterized using the real time RT-PCR. RSV was the predominant virus associated with ARTI with a prevalence of 28% (140/500) in the study sample. RSV in was also detected in more co-infections with other respiratory viruses. RSV was detected throughout the year with peak periods from June to August 2016, March to July 2017 and May to July 2018. Of the 140 RSV positive children tested, 72.14% had RSV-B, while 27.86% had RSV-A infection. Both RSV subtypes were detected throughout the study period with overlapping patterns. A few co-infections between RSV-A and RSV-B were detected during the co-circulation. RSV was the most prevalent virus and RSV-B was the predominant subgroup associated with ARTI in the children <5 years in Sri Lanka from May 2016 to July 2018. RSV was detected throughout the study period with peaks in certain months in the study area.

We evaluated the diagnostic performance of dried blood spots (DBS) compared to plasma for detection and quantification of HBV-DNA under real-life conditions. Blood specimens from 100 known cases of chronic hepatitis B (CHB) requested for quantitative HBV-DNA were included. In a subset of 20 patients, three sets of DBS cards were prepared, one kept at -80°C, the other two kept at room temperature for 7 and 14 days, respectively. DBS method demonstrated sensitivity, specificity, and positive predictive value of 95.3%, 100%, and 100%, respectively in comparison to plasma. The mean HBV-DNA load in plasma was 4.8 log10 IU/ml while in DBS was 4.3 log10 IU/ml with a strong correlation (R²=0.9087). No significant change in viral load was observed at room temperature for up to 14 days. This study suggests that DBS for HBV viral-load quantitation is a good alternative to plasma as it is stable during storage at room temperature and therefore allows easy handling, storage, and transport of specimens in resource-limited settings.

Spring water gargle (SWG) is a suitable, non-invasive, alternative specimen for SARS-CoV-2 detection by RT-PCR. This study sought to evaluate the performance of the cobas Liat point-of-care system for the detection of SARS-CoV-2 in SWG samples. SWG samples and standard oral and nasopharyngeal swab (ONPS) were collected simultaneously from participants in a COVID-19 screening clinic, in November and December 2020. Both sample types were analyzed in parallel on the cobas Liat platform and with the Seegene Allplex 2019-nCoV assay. Among the 110 participants, 53% had compatible symptoms and 71% had a contact with a confirmed COVID-19 case. Only two (1.8%) individuals had neither symptoms nor contact. Amongst 110 paired samples, 25 (23%) were positive for SARS-CoV-2 on the cobas Liat for a least one sample type, with a kappa coefficient of 0.92. Agreement between the cobas Liat platform and the Seegene assay was also excellent (kappa coefficient values of 0.94 and 0.95). Two SWG samples failed to provide a positive result when their ONPS pair was positive, but their cycle threshold (Ct) values were >35 on the Seegene assay, reflecting a low viral load. Overall, the performance of the cobas Liat platform is excellent for the detection of SARS-CoV-2 in SWG samples in a high pre-test probability population.

Early detection of pathogens at the point of care helps reduce the threats to human and animal health from emerging pathogens. Initially, the disease-causing agent will be unknown and needs to be identified; this often requires specific laboratory facilities. Here we describe the development of an unbiased detection assay for RNA and DNA viruses using metagenomic Nanopore sequencing and simple methods that can be transferred into a field setting. Human clinical samples containing the RNA virus SARS-CoV-2 or the DNA viruses human papillomavirus (HPV) and molluscum contagiosum virus (MCV) were used as a test of concept. Firstly, the virus detection potential was optimized by investigating different pretreatments for reducing non-viral nucleic acid components. DNase I pretreatment followed by filtration increased the proportion of SARS-CoV-2 sequenced reads > 500-fold compared with no pretreatments. This was sufficient to achieve virus detection with high confidence and allowed variant identification. Next, we tested individual SARS-CoV-2 samples with various viral loads (measured as CT-values determined by RT-qPCR). Lastly, we tested the assay on clinical samples containing the DNA virus HPV and co-infection with MCV to show the assay's detection potential for DNA viruses.

This protocol is fast (same day results). We hope to apply this method in other settings for point of care detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

The SARS CoV-2 D614G variant circulated in Cuba in 2020. New viral variants were detected after the opening of the border in November 2020. We show the results of the genomic surveillance in Cuba from December 28, 2020, to September 28, 2021 and their relationship to the epidemiological situation in the country. A total of 1,406 nasopharyngeal exudates from COVID-19 patients were processed for RNA extraction and the 1836 bp fragment of the spike gene was amplified and sequenced. The mutations present were determined using the GISAID database. Prevalence ratios were estimated by fitting Poisson univariate and multivariate regression models to investigate associations between SARS-CoV-2 variant group (VOC, non-VOC) and disease outcome. Seventeen genetic variants were detected including VOC Alpha, Beta, Gamma and Delta, one variant of interest (VOI) (Lambda) and two previous VOI (A.2.5.1 and Zeta/P.2). Beta (34.77%), Delta (24.89%) and D614G (19%) variants were the most frequently detected. By June, Delta increased in frequency, displacing Beta. Disease severity increased significantly with age and VOC (PR =1.98, IC 95%: 1.33–3.05, p <0.05). Genomic surveillance allowed us to identify the upsurge of novel variants. Coinciding with the higher epidemic period, multiple variants were co-circulating. Although we cannot rule out that failure in the transmission containment measures occurred, the increase in the number of cases associated with the circulation of several variants, particularly the Beta and Delta variants is highly suggestive. A greater association of Beta variant with clinical severity and Delta variant with a greater transmissibility was observed.

Background

Over 296 million people worldwide are living with chronic Hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established.

Objectives

To develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of serum HBV RNA.

Study design

The 3′ RACE RT-qPCR method was developed using published primers. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed.

Results

The 3′ RACE RT-qPCR assay dynamic range is 25 to 10⁸ copies/µL of synthetic RNA. Theoretical and measured serum HBV RNA quantities from a serially diluted sample showed excellent linearity (R²=0.9795). The inter- and intra-assay repeatability were 94.91% and 95.12%, respectively. Clinical specificity and sensitivity were 100% and 90%, respectively, in treated patients. Inter-laboratory analysis demonstrated moderate to high agreement among participating laboratories (κ = 0.581 to 0.867). High agreement was also observed between both operators participating in the intra-laboratory evaluation (κ = 0.867).

Conclusions

Our methodology for the quantification of serum HBV RNA is specific and repeatable and employs a biologically relevant RNA standard suitable for medium throughput laboratories. Method standardization is required to facilitate the comparison of studies and better understand the clinical role of this novel biomarker.